Study On The Effect And Mechanism Of γ-glutamylcysteine In Attenuating Liver Injury In Mouse Induced By Acetaminophen

Drug induced liver injury refers to liver injury caused by the cytotoxicity of the drug itself or its metabolites when drugs are used in the treatment of diseases.Acetaminophen(APAP)is a widely used antipyretic and analgesic drug.APAP is safe when used at lower than therapeutic doses.However,a single overdose or long-term administration of APAP can cause liver injury,and overdose of APAP is currently the main factor leading to drug induced liver injury.After taking therapeutic dose of APAP,90% of APAP will be non-toxic metabolized through glucuronidation and sulfation reactions,and about 5% of APAP will be metabolized by the cytochrome P450 system into strong electrophilic N-acetyl-pbenzoquinone imine(NAPQI).NAPQI will be metabolized by glutathione(GSH),but a large amount of NAPQI produced by overdose of APAP will deplete GSH quickly.GSH is an important non-protein thiols that maintains the redox balance in cells.In the absence of GSH,highly reactive NAPQI will directly covalent bonding with macromolecules in cells forming protein adducts,and damages the organelles especially mitochondria.Meanwhile,the contents leaked from mitochondria and the oxygen free radicals produced by cell metabolism activities will cause severe oxidative stress due to the insufficient of GSH that eventually lead to cell death.Considering the important role of GSH in metabolizing NAPQI and eliminating free radicals,increasing the content of GSH in liver cells is an effective method to treat APAP poisoning.Since most mammalian cells cannot directly take up complete GSH,and GSH will be rapidly degraded in the digestive tract and blood,the medicinal usage of exogenous GSH is limited.N-Acetyl-L-cysteine(NAC)is currently the only drug approved as an antidote for APAP,which provides cells with the restricted substrate for de novo synthesis of GSH via two steps reactions to restore the GSH content in cells.However,NAC is limited in treatment time and therapeutic effect,and has some uncomfortable side effects.Developing more efficient and safer alternative drugs than NAC has important practical significance in reducing patient suffering and saving patients’ lives.γ-glutamylcysteine(γ-GC)is the direct precursor for the synthesis of GSH via one step reaction.Compared with NAC,γ-GC has the characteristics of higher synthesis efficiency of GSH and no side effects.γ-GC has antioxidant and anti-inflammatory effects,while the role of γ-GC in APAP induced liver injury has not been reported yet.This study aims to evaluate the role of γ-GC in a mouse acute liver injury model induced by APAP overdose.We induced acute liver injury in mice by intraperitoneal injection a single dose of APAP at 400 mg/kg.The experimental results showed that liver GSH decreased by more than 90% after 90 minutes of intraperitoneal injection of APAP.After 12 hours,large areas of necrosis and cell apoptosis occurred around the liver veins of mice.And serum AST/ALT,LDH,HMGB-1 and IL-1β levels are elevated,GSH in the liver continues to be significant reduced,ROS and oxidative stress caused by lipid peroxidation products(MDA and 4-HNE)and protein nitration products(3-NT)are increased.If γ-GC(510 or1020mg/kg/day)is given orally to mice for seven consecutive days before APAP injection,H&E staining shows that liver tissue necrosis caused by APAP is significantly reduced;serological testing shows that serum AST,ALT and LDH caused by APAP were also suppressed;ELISA test showed that APAP induced blood pro-inflammatory factors HMGB-1 and IL-1β levels were significantly suppressed;TUNEL test showed that APAP-induced liver cell apoptosis was significantly reduced.As a precursor of GSH,γ-GC can increase the level of intracellular GSH.Our research results showed that pre-administration of γ-GC to mice inhibits liver GSH deletion induced by APAP,reduces the production of ROS,and reduces MDA,4-HNE and 3-NT levels in liver.If γ-GC(1020mg/kg)is given orally to mice2 hours after APAP injection,γ-GC restores the GSH content in the liver to near normal levels within 2 hours,and reverses APAP-induced liver tissue necrosis and cell apoptosis.And decreases serum AST,ALT,LDH,HMGB-1 and IL-1β levels,increases the level of GSH in the liver,reduces the production of ROS,and decreases MDA,4-HNE and 3-NT levels in liver.In addition,γ-GC also speed up the regeneration and repair process after liver injury via unclear mechanisms.In conclusion,we confirmed that oral γ-GC can reverse the acute liver injury caused by APAP in mice.γ-GC reduces liver tissue necrosis and hepatocyte apoptosis,and reduces inflammatory response and oxidative stress damage caused by APAP through directly synthesizing GSH and reducing ROS production.Our research shows that γ-GC is superior to NAC in reducing liver cell apoptosis and promoting regeneration and repair after liver injury.Considering the advantages of γ-GC in the efficiency and safety of GSH synthesis,we believe that γ-GC has the potential to replace NAC as a APAP antidote.